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Background A novel gradient, high-sensitive and specific stability-indicating reverse-phase HPLC method was developed and validated for quantitative purpose of known, unknown and degradant impurities profiling for diltiazem hydrochloride tablets. The impurities were separated on the Zorbax RX C8 column (150 mm × 4.6 mm, 5 μm) with mobile phase-A consisting of a mixture of 0.05 M sodium dihydrogen phosphate monohydrate buffer pH 3.0 and methanol in the ratio 800:200v/v and mobile phase-B consisting of acetonitrile with a flow rate of 1.0 mL min −1 . The column compartment was maintained at 35 °C, and the detection wavelength was 240 nm. Diltiazem hydrochloride, its known impurities and unknown impurities have been well resolved from each other. Results The linearity of the method has been demonstrated across the concentration range of 0.18 to 5.65 µg mL −1 for EP impurity-F with correlation coefficient R 2 greater than 0.99. Recovery of method was proved from LOQ to 150% for known and unknown impurities with respect to test concentration and found in between 80 and 120%. Forced degradation study and specificity experiment results with mass balance proved the stability-indicating nature of the method and separated all known, unknown impurities and degradants from each other as well as from main drug component (diltiazem hydrochloride). The mass balance for stress study was found in between 95 and 105%. Conclusion Newly developed analytical method was validated as per ICH Q2 (R1) guidelines “Validation of analytical procedure” and found linear, accurate, specific, robust and precise in the established working range.

Background Pediatric leukemias have a diverse genomic landscape associated with complex structural variants, including gene fusions, insertions and deletions, and single nucleotide variants. Routine karyotype and fluorescence in situ hybridization (FISH) techniques lack sensitivity for smaller genomic alternations. Next-generation sequencing (NGS) assays are being increasingly utilized for assessment of these various lesions. However, standard NGS lacks quantitative sensitivity for minimal residual disease (MRD) surveillance due to an inherently high error rate. Methods Primary bone marrow samples from pediatric leukemia ( n  = 32) and adult leukemia subjects ( n  = 5), cell line MV4–11, and an umbilical cord sample were utilized for this study. Samples were sequenced using molecular barcoding with targeted DNA and RNA library enrichment techniques based on anchored multiplexed PCR (AMP®) technology, amplicon based error-corrected sequencing (ECS) or a human cancer transcriptome assay. Computational analyses were performed to quantitatively assess limit of detection (LOD) for various DNA and RNA lesions, which could be systematically used for MRD assays. Results Matched leukemia patient samples were analyzed at three time points; diagnosis, end of induction (EOI), and relapse. Similar to flow cytometry for ALL MRD, the LOD for point mutations by these sequencing strategies was ≥0.001. For DNA structural variants, FLT3 internal tandem duplication (ITD) positive cell line and patient samples showed a LOD of ≥0.001 in addition to previously unknown copy number losses in leukemia genes. ECS in RNA identified multiple novel gene fusions, including a SPANT-ABL gene fusion in an ALL patient, which could have been used to alter therapy. Collectively, ECS for RNA demonstrated a quantitative and complex landscape of RNA molecules with 12% of the molecules representing gene fusions, 12% exon duplications, 8% exon deletions, and 68% with retained introns. Droplet digital PCR validation of ECS-RNA confirmed results to single mRNA molecule quantities. Conclusions Collectively, these assays enable a highly sensitive, comprehensive, and simultaneous analysis of various clonal leukemic mutations, which can be tracked across disease states (diagnosis, EOI, and relapse) with a high degree of sensitivity. The approaches and results presented here highlight the ability to use NGS for MRD tracking.

Dis3 encodes a conserved RNase that degrades or processes all RNA species via an N-terminal PilT N terminus (PIN) domain and C-terminal RNB domain that harbor, respectively, endonuclease activity and 3′–5′ exonuclease activity. In Schizosaccharomyces pombe, dis3 mutations cause chromosome missegregation and failure in mitosis, suggesting dis3 promotes cell division. In humans, apparently hypomorphic dis3 mutations are found recurrently in multiple myeloma, suggesting dis3 opposes cell division. Except for the observation that RNAi-mediated depletion of dis3 function drives larval arrest and reduces tissue growth in Drosophila, the role of dis3 has not been rigorously explored in higher eukaryotic systems. Using the Drosophila system and newly generated dis3 null alleles, we find that absence of dis3 activity inhibits cell division. We uncover a conserved CDK1 phosphorylation site that when phosphorylated inhibits Dis3’s exonuclease, but not endonuclease, activity. Leveraging this information, we show that Dis3’s exonuclease function is required for mitotic cell division: in its absence, cells are delayed in mitosis and exhibit aneuploidy and overcondensed chromosomes. In contrast, we find that modest reduction of dis3 function enhances cell proliferation in the presence of elevated Ras activity, apparently by accelerating cells through G2/M even though each insult by itself delays G2/M. Additionally, we find that dis3 and ras genetically interact in worms and that dis3 can enhance cell proliferation under growth stimulatory conditions in murine B cells. Thus, reduction, but not absence, of dis3 activity can enhance cell proliferation in higher organisms.

BackgroundLeft main disease (LMD) and three-vessel disease (3VD) have important prognostic value in patients with coronary artery disease. However, uncertainties still exist about their prevalence and predictors in patients with acute coronary syndrome (ACS) and also in patients with stable coronary disease. Thus the aim of this study was to perform an international collaborative systematic review and meta-analysis to appraise the prevalence and predictors of LMD and 3VD.MethodsMedline/PubMed were systematically searched for eligible studies published up to 2010, reporting multivariate predictors of LMD or 3VD. Study features, patient characteristics, and prevalence and predictors of LMD and 3VD were abstracted and pooled with random-effect methods (95% CIs).Results17 studies (22 740 patients) were included, 11 focusing on ACS (17 896 patients) and six on stable coronary disease (4844 patients). In the ACS subgroup, LMD or 3VD occurred in 20% (95% CI 7.2% to 33.4%), LMD in 12% (95% CI 10.5% to 13.5%), and 3VD in 25% (95% CI 23.1% to 27.0%). Heart failure at admission and extent of ST-segment elevation in lead aVR on 12-lead ECG were the most powerful predictors of LMD or 3VD. In the stable disease subgroup, LMD or 3VD was found in 36% (95% CI 18.5% to 48.8%), with the most powerful predictors being transient ischaemic dilation during the imaging stress test, extent of ST-segment elevation in aVR and V1 during the stress test, and hyperlipidaemia.ConclusionsThis meta-analysis demonstrated that severe coronary disease—that is, LMD or 3VD—is more common in patients with ACS or stable coronary disease than generally perceived, and that simple and low-cost tools may help in the selection of the most appropriate therapeutic approach.

Existing metrics for grading mitral regurgitation (MR) are limited and fraught with high interobserver variability. We developed and evaluated a Doppler-based, semiquantitative novel index (Mitral Regurgitation Severity Index [MRSI]) of MR severity. In a total of 125 patients (70 in the derivation cohort and 55 in the validation cohort), MRSI was calculated as a ratio of time velocity integral of mitral inflow (continuous-wave Doppler-TVI MV) to the time velocity integral of the left ventricle outflow (pulse-wave Doppler-TVI LVOT). Inter-rater agreement for MRSI and predictive ability of the MRSI were then assessed. In the derivation cohort, MRSI differed significantly between patients with severe MR (2.6 ± 0.51) and mild-moderate (nonsevere) MR (1.4 ± 0.18) and a cutoff of ≥1.8 was associated with optimal diagnostic accuracy. In the validation cohort, MRSI exhibited excellent agreement between a level II and a level III reader with a mean difference of −0.14 (95% confidence limit of agreement: −0.80 to 0.53), correlation coefficient of 0.88 (p <0.001), and 16% CV; and using the cut point of 1.8, it exhibited good inter-rater reproducibility with a kappa coefficient of 0.72 (p <0.001). In conclusion, MRSI appears to be a simple, quantitative, practical, color-independent metric to differentiate severe MR from nonsevere MR.

The orphan small nucleolar RNA (snoRNA) ACA11 is overexpressed as a result of the t(4;14) chromosomal translocation in multiple myeloma (MM), increases reactive oxygen species, and drives cell proliferation. Like other snoRNAs, ACA11 is predominantly localized to a sub‐nuclear organelle, the nucleolus. We hypothesized that increased ACA11 expression would increase ribosome biogenesis and protein synthesis. We found that ACA11 overexpression in MM cells increased nucleolar area and number as well as argyrophilic nuclear organizing regions (AgNORs). Supporting these data, samples from t(4;14)‐positive patients had higher AgNORs scores than t(4;14)‐negative samples. ACA11 also upregulated ribosome production, pre‐47S rRNA synthesis, and protein synthesis in a ROS‐dependent manner. Lastly, ACA11 overexpression enhanced the response to proteasome inhibitor in MM cells, while no effect was found in response to high doses of melphalan. Together, these data demonstrate that ACA11 stimulates ribosome biogenesis and influences responses to chemotherapy. ACA11 may be a useful target to individualize the treatment for t(4;14)‐positive myeloma patients.

The current definition of peripartum cardiomyopathy (PC) is restricted to patients with left ventricular systolic dysfunction (ejection fraction [EF] <45%). Data on peripartum heart failure (HF) with normal EF are sparse. We describe clinical characteristics of patients with normal (≥55%) and patients with low (<45%) left ventricular ejection fractions (LVEFs). Electronic medical records (2006 to 2013) of our tertiary care center were retrospectively screened to identify peripartum HF with normal EF, defined as an entity meeting Framingham criteria for HF with symptom onset during the last month of pregnancy or up to 5 months after delivery and with an EF of ≥55%. Clinical characteristics, echocardiographic parameters, and outcomes of these patients were compared with age-matched control patients with traditionally defined PC (EF <45%). A total of 25 patients with PC and EF ≥55% were identified. Exclusion of hypertension (n = 9), preeclampsia (n = 1), and diabetes mellitus (n = 2) yielded 13 patients with PC and EF ≥55%. Age-matched patients with traditional PC (EF <45%) constituted controls (n = 16). Compared with patients with PC and low LVEF, patients with PC and normal LVEF had lower B-type natriuretic peptide levels, systolic and diastolic left ventricular dimensions, left atrial size, and incidence of decompensated HF during delivery (p <0.05). Compared with historical age-matched controls, patients with normal LVEF exhibited attenuated E′ mitral annular velocities. On follow-up, these patients were associated with a lower New York Heart Association functional class. In conclusion, peripartum HF with normal LVEF appears to be a distinct entity.

Streptococcus pyogenes, or group A Streptococcus (GAS), a gram-positive bacterium, is implicated in a wide range of clinical manifestations and life-threatening diseases. One of the key virulence factors of GAS is streptopain, a C10 family cysteine peptidase. Since its discovery, various homologs of streptopain have been reported from other bacterial species. With the increased affordability of sequencing, a significant increase in the number of potential C10 family-like sequences in the public databases is anticipated, posing a challenge in classifying such sequences. Sequence-similarity-based tools are the methods of choice to identify such streptopain-like sequences. However, these methods depend on some level of sequence similarity between the existing C10 family and the target sequences. Therefore, in this work, we propose a novel predictor, C10Pred, for the prediction of C10 peptidases using sequence-derived optimal features. C10Pred is a support vector machine (SVM) based model which is efficient in predicting C10 enzymes with an overall accuracy of 92.7% and Matthews’ correlation coefficient (MCC) value of 0.855 when tested on an independent dataset. We anticipate that C10Pred will serve as a handy tool to classify novel streptopain-like proteins belonging to the C10 family and offer essential information.

The role of CRP as a mediator in atherosclerosis and inflammation is being investigated worldwide. In the present study, the effect of CRP on matrix metalloproteinases (MMP)-1, 2, 9, and their tissue inhibitor (TIMP-1) gene expression in THP-1 monocytic cell line was investigated. Specific mitogen activated protein (MAP) kinase (ERK, p38, and JNK) inhibitors were used to elucidate the signaling pathways involved. Effect of atorvastatin was determined in the presence of CRP on the expression of genes. Time and dose-dependent experiments were performed in the presence of CRP. The results showed that the treatment of THP-1 cells with 100 μg of CRP/ml/10⁶ cells for 24 h enhanced the expression of MMPs and TIMP-1 genes significantly. CRP upregulated the expression of these genes via FcγRII and utilized ERK signaling pathway to transduce signals. Atorvastatin was able to significantly attenuate CRP-induced MMPs expression and augmented TIMP-1 gene expression significantly. In conclusion, CRP is not only a risk marker for vascular events, but also directly involved in the mechanisms leading to remodeling and destabilization of atherosclerotic plaque. Also, atorvastatin serves as potential therapeutic modality to curb these harmful events.